Glioblastoma multiforme (GBM) is the most lethal form of brain cancer in adults. Despite a recent explosion of information on the genetic causes of this disease, the average survival of GBM patients is only 15 months and the tumors tend to be resistant to all known therapies. The devastating nature of GBM underscores a dire need for novel therapeutic targets and a better understanding of the molecular mechanisms that drive the growth and maintenance of these tumors. Like other aggressive tumors, GBMs display a high degree of cell proliferation made possible by the activation of a select set of metabolic activities required to generate the energy and macromolecules (lipids, nucleic acids, proteins) needed for cell growth and division. These metabolic activities are attractive therapeutic targets because they lie between the diverse molecular drivers of cellular transformation in GBM and the phenotype of rapid tumor growth that ultimately kills the patient. The main objective of this proposal is to define the metabolic regulators of GBM cell growth in vitro and in vivo. The proposal builds on our novel analytical methods developed to measure metabolism of GBM cells in culture and, for the first time, in intact human GBM tumors grown in the mouse. These methods led to the surprising observation that GBM cells have highly active mitochondria that use either of two metabolic pathways to convert nutrients into precursors required for macromolecular synthesis and growth. Specific Aim 1 proposes loss-of-function experiments with RNA interference to identify key metabolic regulators that allow GBM cells to use the amino acid glutamine as the preferred mitochondrial substrate. Specific Aim 2 proposes similar experiments to define the key metabolic regulators in an alternative metabolic pathway that does not require glutamine but still allows tumor cells to grow rapidly in culture and in vivo. Specific Aim 3 proposes to study metabolism in twenty independent human GBM tumors grown exclusively in the mouse brain to understand how tumor mutations influence metabolic activity, and whether particular mutations define sensitivity to the suppression of metabolic activities in vivo. All three Specific Aims feature extensive metabolic analysis of live tumors using nuclear magnetic resonance spectroscopy and mass spectrometry, and will produce a unique view of GBM metabolism with an unprecedented level of detail and biological accuracy. Together, these experiments will pinpoint a new set of metabolic activities that distinguish GBM tumors from normal brain, will define mechanisms that connect malignant transformation to cellular metabolism, and will identify metabolic targets that should lead to novel therapeutic approaches in GBM and potentially other aggressive cancers.